Primers and probes for amplification and detection of human inhibitor of DNA-binding

ABSTRACT

Oligonucleotide primers and polynucleotide probes provide selective amplification and detection of Id genetic sequences and are selected both (1) to provide the desired specificity so that they amplify only the specific Id type to which they are targeted; and (2) to introduce terminal restriction endonuclease cleavage sites into the amplicon that facilitate the incorporation of the amplicon into plasmid-based vectors. Detection of Id genetic sequences can be carried out using the labeled-amplicon by reamplifying the amplicon with the primers in the presence of labeled, for example radiolabeled, deoxynucleotide triphosphates. The probes of the invention correspond in sequence to the amplicons produced using the primers of the invention, after cleavage at the restriction endonuclease sites.

This application claims the benefit of U.S. Provisional Application No.60/475,515, filed Jun. 3, 2003, which application is incorporated hereinby reference for purposes of the US filing and all other jurisdictionspermitting such incorporation.

The invention was supported with funds from NSF Grant No. IBN-9118977.The United States government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

This application relates to oligonucleotide primers and nucleic acidprobes that are useful for amplification and/or detection of humaninhibitor of DNA-binding (Id) genetic sequences, and in particular forselective amplification and/or detection of human Id1, Id2 and Id3.

Inhibitor of DNA-binding (Id) proteins are transcription factors and aremembers of a subfamily of Helix-Loop-Helix (HLH) proteins. Theseproteins contain a motif that consists of two segments capable offorming amphipathic alpha helices connected by a nonconserved loop.Other members of the HLH family (basic HLH, or bHLH proteins) alsocontain a basic region just to the amino terminal side of this motifthat consists of two to three clusters of basic amino acid residues.Various proteins containing the bHLH motif can form homodimeric andheterodimeric complexes with other bHLH proteins and it is through thebasic region that these complexes bind to the target DNA (Murre et al.,Cell, 1989, 56, 777-783; Murre et al., Cell, 1989, 58, 537-544). The Idproteins containing the helix-loop-helix domain, but are lacking thebasic region. These Id proteins are still able to form heterodimers withother bHLH transcription factors affecting transcription, but they lackDNA-binding ability and are therefore negative regulators of the bHLHtranscription factors.

Four members of the Id family have been identified in mammals and thefirst, Inhibitor of DNA binding-1 (Id-1), originally isolated in themouse, has been shown to exist in two forms in the human as a result ofalternative splicing (Benezra et al., Cell, 1990, 61, 49-59; Deed etal., Biochim. Biophys. Acta., 1994, 1219, 160-162; Hara et al., J. Biol.Chem., 1994, 269, 2139-2145; Nehlin et al., Biochem. Biophys. Res.Commun., 1997, 231, 628-634; Zhu et al., Brain Res. Mol. Brain Res.,1995, 30, 312-326).

SUMMARY OF THE INVENTION

The present invention provides oligonucleotide primers andpolynucleotide probes that provide selective amplification and detectionof Id genetic sequences. Forward and reverse primer pairs foramplification of Id1, Id2 and Id3 are given in Seq. ID Nos 1 and 2, 3and 4 and 5 and 6 respectively. These primer pairs were selected both(1) to provide the desired specificity so that they amplify only thespecific Id type to which they are targeted; and (2) to introduceterminal restriction endonuclease cleavage sites into the amplicon thatfacilitate the incorporation of the amplicon into plasmid-based vectors.Detection of Id genetic sequences can be carried out using thelabeled-amplicon by reamplifying the amplicon with the primers in thepresence of labeled, for example radiolabeled, deoxynucleotidetriphosphates. The probes of the invention correspond in sequence to theamplicon produced using the primers of the invention, after cleavage atthe restriction endonuclease sites. The sequences of sense probes thatcorrespond to wild-type human Id1, Id2, and Id3 are set forth in Seq IDNos. 7, 8 and 9 respectively. Antisense strands of the same sequence mayalso be employed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to two types of nucleic acid polymerswhich are useful in the detection of human Id1, Id2 or Id3 geneticsequences. For convenience, these two types of nucleic acid polymers arereferred to herein as “primers” and “probes” although it will beappreciated from the description below that the shorter “primers” canalso be used in detection procedures, and that the longer “probes” canalso serve as a primer for extension reactions. Thus, the names are usedas labels for clarity, and there is no implication in the name that theutility of the nucleic acid polymer is in any way limited to the singlefunction of the name.

As used in the specification and claims of this application, the term“genetic sequences” refers to either DNA or RNA sequences encoding someor all of human Id1, Id2 or Id3 protein, both in vivo and in vitro. The“genetic sequences” may be amplification products or they may beunamplified materials. The “genetic sequence” may be detected in situ toobtain maps of Id protein expression, it may be detected in a milieu ofother human nucleic acids and cellular components, or it may be detectedin an artificial environment, such as in host cells (prokaryotic oreukaryotic) expressing a plasmid-based vector harboring nucleic acidsencoding a human Id protein.

The primers of the present invention were developed to meet severalspecific goals. First, each set of forward and reverse primers wasdesigned to have little or no cross-reactivity with other Id proteinsand members of the HLH family in hybridization experiments with humantissue samples. Second, the primers each include a restrictionendonuclease cleavage site, such that the ends can be trimmed fromamplicons produced using the primers in a PCR amplification to produce asequence that can be readily inserted into a plasmid-based vector forcloning to produce multiple copies of the amplicon for use as a probe.Construction of oligonucleotides of defined sequence is routine andcompanies exist to provide requested materials. Primers of the inventioncan be constructed using such known techniques for synthesis ofoligonucleotides.

For Id1, the primers of the invention have the sequence:

Forward: ataggatccc accctcaacg gcgagat Seq ID No. 1 Reverse:gtggaattcc ccacagagca cgtaattcct Seq ID No. 2In each sequence, the underlined portions corresponds to the sequence ofId1, while the remainder is an introduced segment to provide therestriction endonuclease cleavage site.

For Id2, the primers of the invention have the sequence:

Forward: ataggatccc cgcatcccac tattgtca Seq ID No. 3 Reverse:gtggaattca acaccgtcta ttcagccaca Seq ID No. 4In each sequence, the underlined portions corresponds to the sequence ofId2, while the remainder is an introduced segment to provide therestriction endonuclease cleavage site.

For Id3, the primers of the invention have the sequence:

Forward: ataggatcca ccttcccatc cagacagcc Seq ID No. 5 Reverse:gtggaattcc ctgagcacca ggttcagtct Seq ID No. 6In each sequence, the underlined portions corresponds to the sequence ofId3, while the remainder is an introduced segment to provide therestriction endonuclease cleavage site.

These primers pairs can be used for selective amplification of Id1, Id2or Id3 sequences using polymerase chain reaction procedures. In thiscase, the amplification products are suitably separated by size on amatrix such as a polyacrylamide gel and incorporated label detected.Suitable labels include without limitation radio-labels, fluorescentlabels, colored labels, and fluorogenic or chromogenic labels.

The amplicons of sizes characteristic of the Id1, Id2 and Id3 genes arealso used in accordance with the invention for construction of vectorswhich can be used in the production of the probes of the invention. Theamplicons are purified (for example by purification-scaleelectrophoresis), digested with restriction endonucleases BamH1 andEcoR1 and cloned into a BamH1/EcoR1 site of a vector, such as apBluescript vector (pBS-KS-). The modified vector is introduced into asuitable host, for example E. coli in the case of pBluescript, to makemultiple copies of the vector. These copies are recovered, and the probeof the invention obtained by direct reamplification of the plasmid.

The probes of the invention have lengths of 147 bp, 171 bp and 241 bpfor Id1, Id2 and Id3, respectively, and the sequences as set forth inSeq. ID. Nos. 7, 8 and 9. The probes of the invention are suitably usedin Northern hybridization analysis of RNA extracted from human cellsthat are to be assessed for the expression of one or more Id proteins.In a general sense, the probes of the invention can be used forqualitative or semi-quantitative assessment of Id mRNA levels in humantumor specimens. The specificity exhibited by these probes makes them ofgreat utility in distinguishing between different Id family members, aswell as between Id and other members of the helix-loop-helix family.Such measurements have diagnostic and prognostic value in the managementof human disease.

For diagnostics, it has been observed that there are characteristicpatterns of Id expression which distinguish some tumor types fromcorresponding normal tissues. For example, Northern analysis using theprobes of the invention can be used to distinguish humanrhabdomyosarcoma cells from normal primary human muscle cells. (SeeInternational Patent Publication WO97/05283 (designating the US), whichis incorporated herein by reference in all jurisdictions permitting suchincorporation.). Another, non-limiting example of the use to which theprobes of the invention may be put is the evaluation of human braintissue samples for the presence of astrocytomas. Deregulated Idexpression has also been observed in disseminated medullablastoma, stageII/IV neuroblastoma and melanoma.

For prognostic purposes it has been observed that the presence of Id2 inneuroblastoma of children is an indicator of poor prognosis. Thus,detection of Id2 using the probes of the invention can be used as abasis for selecting a more aggressive course of therapy and for enhancedmonitoring of the individual for disease recurrence. Similarly, Id1positivity in astrocytomas and early stage lesions of the skin have beenshown to signal more aggressive disease, and changes in diseasetreatment and management may therefore be indicated.

The probes of the invention may also be used in detection of low levelsof Id in circulating tumor cells as a measure of metastatic risk incertain patient populations. High Id levels have also been observed incirculating endothelial cell precursors which are required forvascularization of certain tumors and which may be the targets ofanti-angiogenic drug regimens. Monitoring of Id levels in these cellsmay be used for determining if a tumor is recruiting new blood vesselsand therefore likely to begin metastasizing and whether angiogenicintervention is having the desired effect.

EXAMPLE 1

A PCR reaction was performed using human Id1 or human genomic DNAtemplate and an Id1-specific primer pair (Seq. ID Nos. 1 and 2). Eachreaction contained 5 ng of template, 500 ng of each of the 5′ and 3′primers, 5 μl of 10× PCR mix (500 mM KCl, 100 mM Tris HCl pH 8.4, 15 mMMgCl₂, 1 mg/ml gelatin), 20 μM of each deoxynucleotide triphosphate and3 units of Taq polymerase in a 50 μl reaction volume. PCR was performedfor 25 cycles to produce amplicons using a standard Perkin-Elmerautomated PCR machine (60° C. hybridization temperature, 72° C.extension temperature). The amplification mixtures were loaded on apolyacrylamide gel and separated by electrophoresis. The product bandwas excised, and the resulting gel slices were rotated at roomtemperature for 2 days in elution buffer (0.3 M sodium acetate, pH 8.0,5 mM EDTA). Supernatant was removed from the rotated slices, and theslices were rinsed again in 100 μl elution buffer. The removedsupernatant and the elution buffer from the rinse were pooled in to anEppendorff tube to yield a total volume of about 400 μl. 40 μl 5M NaCl,4 μl 1M MgCl₂ and 600 μl isopropyl alcohol were added to the tube whichwas centrifuged for 30 minutes. The resulting pellet was washed in 80%ethanol and dried under vacuum for 5 minutes. The pellet was thenresuspended in 15 μl PCR sterile TE to form an amplicon solution.

The probe can now be used directly used directly to make a probe for insitu hybridization by reamplification as described above using³²P-labeled deoxynucleotide triphosphates. The reamplified, labeledprobe is separated from unincorporated dNTPs using a G-50 Sephadex spincolumn and applied to tissue samples as described in Jen et al.

The unlabeled probe was also cloned into the BamHI+EcoRI sites ofpBluescript (KS⁻) (Stratagene) to ensure a limitless supply of templatewithout reamplification of cDNA. 10 μl of the unlabeled ampliconsolution was combined with 3 μl of 10× KGB buffer, 8 units of of BamH1,8 units of EcoR1, and 15.5 μl of water and incubated at 37° C. for about4 hours to cleave the BamH1 and EcoR1 restriction sites. 40 μl TE (10 mMTris pH 7.4, 1 mM EDTA) and 40 μl phenol (pH 8.0) was added to the mix,centrifuged to recover the aqueous phase which was then applied to aG-50 Sepharose column to remove residual phenol. The material wasprecipitated by adding 1/10 volume of 3 M NaOAc, EtOH and centrifugedfor 30 minutes.

The recovered pellet was resuspended in 10 μl PCR-sterile TE. 3 μl ofthis suspension was combined with BamH1/EcoR1 cut pBluescript vector(pBS-KS⁻), ATP and ligase in buffered and incubated overnight at 15° C.The ligated vector was transferred into E. coli strain JM109 usingstandard protocols. After growth, colonies harboring the ampicillinresistance marker of the Bluescript vector were selected by plating onampicillin containing agar. Plasmids were isolated and tested for thepresence of the amplicon insert. The resulting probe has the sequencegiven in Seq. ID No. 7.

EXAMPLE 2

A probe was created for human Id2 using an Id2-specific primer pair(Seq. ID Nos. 3 and 4) in the procedures of Example 1. The resultingprobe has the sequence given in Seq. ID No. 8.

EXAMPLE 3

A probe was created for human Id3 using an Id3-specific primer pair(Seq. ID Nos. 5 and 6) in the procedures of Example 1. The resultingprobe has the sequence given in Seq. ID No. 9.

EXAMPLE 4

RNA was extracted from human rhabdosarcoma cancer cell lines and primaryhuman muscle cells (as a control). Northern analysis was performed usingthe procedures previously described in Benezra et al. (1990) Cell 61:49-59, which is incorporated herein by reference, and the Id1, Id2 andId3 probes (Seq. ID Nos 7-9). The observed hybridization patternsdemonstrated the specificity of the probes, since the Id1 probehybridized to a differently and correctly sized messenger, as comparedto Id2 and Id3. In addition, in case of Id2, the specificity of theprobe was confirmed by hybridization to an Id2 containing plasmid.

EXAMPLE 5

ID1 specific probe (Seq. ID No. 7) is used for in situ hybridization inthe evaluation of sample suspected of being early stage melanoma. 6-7micron sections are processed with [α-³³P]-labeled probes using thegeneral procedure previously described in Lyden et al., Nature (Lond)401: 670-677 (1999), which is incorporated herein by reference, andobserved for binding of the label. Binding is indicative that the sampleis early stage melanoma.

1. A composition comprising a first primer consisting of SEQ ID NO: 1and a second primer consisting of SEQ ID NO:
 2. 2. A compositioncomprising a first primer consisting of SEQ ID NO: 3 and a second primerconsisting of SEQ ID NO:
 4. 3. A composition comprising a first primerconsisting of a SEQ ID NO: 5 and a second primer consisting of a SEQ IDNO: 6.